Heparin is a highly sulfated, microheterogeneous and polydisperse polysaccharide comprising repeating disaccharide units composed of uronic acid (L-iduronic acid, IdoA or D-glucuronic acid, GlcA) and glucosamine (α-D-glucosamine, GlacN). It has good anticoagulant and antithrombotic activities and thereby is clinically used to prevent venous thrombosis after operation. Enoxaparin sodium represents a low molecular weight heparin, which is obtained by esterifying heparin extracted from intestinal mucosa in pigs to give benzyl ester derivatives of heparin sodium, and then derived from heparin by alkaline degradation. Compared to other heparins, enoxaparin sodium is more complicated in composition because of structural alterations (such as the difference in sulfation sites and numbers) induced by chemical manufacturing procedures. The weight-average molecular weight of enoxaparin sodium is ranging from 3,800 to 5,000 Da; wherein approximately 20% oligosaccharides have a molecular weight of less than 2,000 Da; more than 68% oligosaccharides have a molecular weight between 2,000 and 8,000 Da; and no more than 18% oligosaccharides have a molecular weight of higher than 8,000 Da.
During the manufacturing process, alkaline degradation undergoes two main competitive chemical reactions, namely, β-elimination and hydrolysis of benzyl ester. After degradation, a low molecular weight heparin is obtained in which oligosaccharide chain having an average molecular weight of about 4,500 (U.S. Pat. No. 5,389,618). The resulting oligosaccharide chains of enoxaparin sodium still bear the pentasaccharide structure which displays similar anticoagulant activity present in the parent heparin polysaccharide chains, and such a pentasaccharide sequence accounts for 15-25% in enoxaparin sodium.
During the process of restrictive degradation of heparin, desulfation and deamination may occur, and the glucosamine part at the reducing end of oligosaccharide may undergo the following characteristic conversions: (1) epimerization between glucosamine and mannosamine (T. Toida et al., J. Carbohydrate. Chem. 15(13), 351-360 (1996)), and (2) 6-O-desulfation of 6-O-sulfated glucosamine, to form a structure called 1,6-anhydro ring. These reactions enhance the structural complexity and diversity of enoxaparin sodium. Besides the above mentioned conversions, structural alterations also occur in sugar chain length, sequence and fine structure of building blocks.
The 1,6-anhydro structure at the reducing end of oligosaccharide is a characteristic structure of enoxaparin sodium. The ratio of 1,6-anhydro ring structure refers to the molar percentage of oligosaccharide chains with 1,6-anhydro ring structure. The ratio of 1,6-anhydro ring structure has been used as a criterion in pharmaceutical quality control of enoxaparin sodium as required by the United States Pharmacopoeia and European Pharmacopoeia. According to the European Pharmacopoeia, oligosaccharide chains with 1,6-anhydro ring structure should account for 15-25% of the total oligosaccharide chains.
However, the highly complex structure of enoxaparin sodium (such as the high non-uniformity and difference in the degree of sulfation of disaccharide unit) makes the analysis of its fine structure very difficult.
Strong anion exchange high performance liquid chromatography (SAX-HPLC) is the first choice in analyzing the sulfated oligosaccharide components of enoxaparin sodium. In addition, high performance liquid chromatography or low pressure gel permeation chromatography (GPC) is an effective tool for separating polysaccharide and desalting based on the molecular weight. Chromatographic methods for analysis of completely enzymatic digested samples of enoxaparin have been reported in many literatures (for example, CN03822562.X and CN200580009444.0). Nevertheless, when determined by strong anion exchange chromatography (SAX), several disaccharides cannot be baseline resolved, the α and β anomers at the reducing end of oligosaccharide must be eliminated by reduction with sodium borohydride to avoid the peak split.
Alternatively, capillary electrophoresis (CE) has been increasingly used to analyze sulfated polysaccharides (cf. U.S. Pat. No. 7,575,886 B2, Ampofo, S. et al., Anal. Biochem. 199:249-255 (1991); Malsch et al., J. Chromatogr. A. 716:258-268 (1995)). However, the method for separating and determining the ratio of 1,6-anhyro ring structure formation by capillary electrophoresis has never been reported.
Matrix assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF-MS), which does not require the steps of chromatography, can also be used for the analysis of heparin, and it has be used to sequence oligosaccharide chains (H. Sakaguchi et al., J. Biochem. 129 (2001) 107-118; A. J. Rhomberg, et al., Proc. Nalt. Acad. Sci. USA 95 (1998) 4176-4181; L. Sturiale, et al., Semin. Thromb. Hemost. 27 (2001) 465-472). However, MALDI-TOF-MS is not suitable for analyzing the sample with complex component such as the intact enoxaparin, and is not suitable used for controlling product quality owing to its high cost.